10 muthukumar aug manuscript

8/17/2019 10 Muthukumar Aug Manuscript

1/5

J. Acad. Indus. Res. Vol. 1(3) August 2012 148

©Youth Education and Research Trust (YERT) Muthu Kumar et al., 2012

ISSN: 2278-5213

Antiox idant and ant icancer activi ty of Helicteres isora dr ied fruit solvent extracts

T. Muthu kumar 1, A. Mary Violet Christy1, RCS. Ramya2, M. Malaisamy3, C. Sivaraj3, P. Arjun2,N. Raaman

3 and K. Balasubramanian

2

1Dept. of Biotechnology, Thanthai Hans Roever College, Perambalur-621212;

2Plant Tissue Culture, Food safety and

Microbiology lab, National Agro Foundation, Anna University, Taramani Campus, Taramani, Chennai-600113;3Fungal Biotechnology, Natural Products and Plant Tissue Culture Laboratory, Centre for Advanced Studies in Botany,

University of Madras, Guindy Campus, Chennai-600025. [email protected]; +91 9003032704 ______________________________________________________________________________________________

Abstrac t

Helicteres isora dried fruit solvent extracts were evaluated for their antioxidant and anticancer activity. Acetone fruit extract H. isora showed (96.44%) strong antioxidant activity compared to hexane, andiso-propyl alcohol (IPA). Acetone extract exhibited better cytotoxicity against human lung cancer cells

(NCI-H460) whereas; acetone and crude protein extracts showed activity against reactive oxygen species.Keywords: Helicteres isora, antioxidant, anticancer, acetone, cytotoxicity, reactive oxygen species.

IntroductionHelicteres isora Linn. is a shrub belongs to the familySterculiaceae commonly known as ‘East Indian Screwtree. The plant genus consists of 45 species distributedin warmer regions of hemispheres; four species arereported to occur in India (Anonymous, 1997). They area rich source of medicinal compounds with a wide rangeof properties (Bapalal, 1982), which is also known as Avartani in Ayurveda (Yoganarasimhan, 1996). Tribals ofWyanad, Malappuram and Palghat districts of Kerala,

India, used H. isora plant extracts for its anticancerproperties (Mathew and Unnithan, 1992). Traditionally,the root juice and bark of H. isora were used againstemphysema and diabetes. It is also used as expectorant,astringent, antigalactagogue, to reduce gripping and acure for snakebite (Kirtikar and Basu, 1993, Singh et al.,1984). Helicteres isora fruits are used as astringent,stomachic, vermifuge, vulnerary and useful in bowelgripes (Chopra et al., 1956). Cell cycle inhibitory activityof H. isora against tsFT210 cell line was reported byQuinchun et al. (2005). The present study was aimed toassess the antioxidant and anticancer properties of driedfruit extracts of H. isora considering the above facts.

Materials and methodsChemicals: One percent phosphate buffer saline (PBS),fluorescent probe, 2',7'-dichlorfluorescein-diacetate(DCFH-DA), hexane, acetone, isopropyl alcohol (IPA),coomassie brilliant blue G 250 (CBB-G250), ethanol,ortho-phosphoric acid, bovine serum albumin (BSA),mono and dibasic sodium phosphate, phosphate buffer6.8, 7.2. Analytical grade chemicals supplied by Loba,Hi-Media, S.D. Fine Chemicals, Merck, Qualigens andSigma Chemicals (U.S.A) were used.

Source of plant material: Helicteres isora fruits werecollected during June 2011 to Nov 2011 (Fig. 1).Subsequently, the fresh fruit was collected and washed

thoroughly in running tap water followed by distilledwater. It was then dried at room temperature andpowdered using mechanical pulveriser and subjected forextraction.

Preparation of dried fruit solvent extracts

Acetone extracts were extracted by soaking 250 gpowder in 900 mL of acetone for 72 h. The extract wasfiltered through Whatman No. 40. The above preparationwas repeated thrice. Isopropyl alcohol extracts were

prepared by soaking 250 g powder in 750 mL of IPA for72 h. The extract was filtered through Whatman No. 40,The above preparation were repeated thrice. Hexaneextracts were prepared by soaking 300 g powder in 750mL of hexane for 72 h. The extract was filtered throughWhatman No. 40, the above preparation were repeatedthrice. Acetone, isopropyl alcohol and hexane extractswas condensed in rotary evaporator with constanttemperature at 45ºC at 200 rpm and the residue wasused for further analysis.

RESEARCH ARTICLE


2/5



Extraction and estimation of proteinProtein was extracted using Glass Distilled water andphosphate buffer (pH 6.8 and 7.2). One gram of samplewas grinded with 10 mL of respective solvent.Homogenates and sample was filtered and the filtratewas centrifuged at 5000 rpm for 5 min. supernatant was

treated with ice cold acetone at 4°C, centrifuged at10000 rpm for 10-15 min. supernatant was discardedand pellet was dissolved in respective solvent. Thedissolved protein was estimated (Bradford, 1976). Thesamples were prepared in 100 mL of PBS. Dye bindingsolution (5 mL) was added in each tube, mixed for 5 minwhere red dye turns into blue due to protein binding andabsorbance was read at 595 nm.

Dot plot rapid assayThe rapid screening assay was performed by the methodproposed by Soler-Rivas et al. (2000) with slightmodification. Aliquots of plant extracts (3 μL) were

spotted carefully on TLC plates and dried for 3 min. Thesheets bearing the dry spots were placed upside downfor 10 sec in 0.4 mM DPPH solution and the layer wasdried. The stained silica layer revealed a purplebackground with yellow spots, which showed radicalscavenging capacity.

DPPH free radical scavenging activity

The odd electron in the DPPH free radical gives a strongabsorption maximum at 517 nm and is purple in color.The color turns from purple to yellow as the molarabsorptivity of the DPPH radical at 517 nm reduces from9660 to 1640 when the odd electron of DPPH radical

becomes paired with hydrogen from a free radicalscavenging antioxidant to form the reduced DPPH-H(Nagarajan et al., 2012). The resulting decolorization isstoichiometric with respect to number of electronscaptured. Antioxidant compounds may be water-soluble,lipid soluble, insoluble or bound to cell walls. Hence,extraction efficiency is an important factor inquantification of antioxidant activity of foods. Thereaction mixture consists 1 mL of 0.1 mM DPPH inethanol with various concentrations (500 µg/mL, 250 µg/mL, 125 µg/mL, 64 µg/mL, 32 µg/mL, 16 µg/mL and 8µg/mL). 1 mL of DPPH and 1 mL of ethanol were usedas control and ascorbic acid was used as standard. The

mixture was then allowed to react at room temperaturefor 30 min. Methanol served as the blank and DPPH inmethanol, without the extracts, served as the positivecontrol. After 30 min of incubation, the discolouration ofthe purple colour was measured at 517 nm in aspectrophotometer (Genesys 10-S, USA). The radicalscavenging activity was calculated as follows:% Inhibition = OD control-OD sample/ OD control ×100The 50% inhibitory concentration (IC50) values werecalculated by plotting an x, y scatter trend line withregression equation.

Cytotoxicity determination by MTT assay

Helicteres isora fruit extracts were evaluated foranticancer activity. For the Cytotoxicity/anticancer assay,

NCI-H460 human lung cancer cells were used. Cellswere maintained in DMEM (Dulbeccos modified eaglesmedium) supplemented with FBS (foetal bovine serum)and penicillin/streptomycin-L-glutamine and cultured in ahumified atmosphere of 5% CO2 and 95% air at 37°C inThermo Hera Cell 150 incubator. Cells were seeded in

96 well plates at the density of 5000 cells/well in 100 μLof RMPI 1640 medium. Then various concentrations (10µg/mL, 20 µg/mL, 30 µg/mL and 40 µg/mL) of the crudeextracts and protein samples were added to the cells.Cells were incubated for 24 h with test extractconcentrations (Arjun et al., 2012). Each concentrationwas tested in triplicate.

Cell viability test

MTT assay was used to determine cell viability. MTTassay measures changes in colour for measuring theactivity of enzyme that reduce MTT to formazan, giving apurple colour. Yellow MTT (a tetrazole) reduce to purple

formazan in living cells (Mosmann, 1983). After 24 hincubation, 10 μL of MTT was added to each well andincubated for additional 4 h. Then 100 μL of DMSOsolution was added to each well to solubilize theformazan crystals. The plates were read for opticaldensity at 570 nm using a plate reader. By using opticaldensity, the percentage inhibition of NCI-H460 humanlung cancer cells was calculated. The percentageviability was calculated as follows:

Cell viability = OD of samples/OD of control × 100.

Measurement of intracellular ROS by Fluorescent probe,

2',7'-dichlorfluorescein-diacetateCell suspension (0.2 mL) was taken (1X10

6) and

incubated with the test sample. Cells (200 mL) weretaken after treatment and diluted in 3 mL of PBS and 300μL DCFH-DA (1 μg/mL) was added. Reading was takenimmediately without any delay using spectrofluorometer(excitation-485 nm, emission–530 nm) and incubated for30 min in dark conditions (Bhosle et al., 2005).

Fluorescence microscopy for ROS measurement

Test compound was added to suspend cells (1x106

cells/mL) in 1 mL warm medium in small petri dishes. After the treatment, DCFH-DA dye (1 μg/mL) was added

and incubated at 37ºC for 30 min. Then, the cells werewashed once by adding 2 mL of warm PBS and finallythe cells were viewed under fluorescence microscopeusing blue filter (Muthu kumar et al., 2012).

Assessment of mitochondrial membrane potential

Approximately 1x106 cells/mL was added in 1 mL

medium in dark test tubes and the treated sample wasadded and incubated at different time intervals (24/48/72h). 10 μL Rhodamine-123 dye (10 μg/mL) was addedand cells were incubated at 37ºC in 5% CO2 for 30 minand centrifuged at 1200 rpm for 10 min. Supernatant wasdiscarded and 2 mL of warm PBS was added in pellet.

Finally it was read at 480 and 550 nm usingspectrofluorometer.


3/5



Fluorescence microscopy method

Approximately 1x106

cells/mL was added in 1 mLmedium in petri dishes and treated sample was addedand incubated. 10 μL Rhodamine-123 dye (10 μg/mL)was added and incubated at 37ºC with 5% CO2 for 30min and washed with 2 mL of warm PBS. Finally the cellswere observed under fluorescence microscope (510-590

nm).

Analysis of apoptotic morphological changes Approximately 1x10

6 cells/mL was added in 1 mL

medium in petri dishes and treated sample was addedand incubated. Fifty μL/mL EB/AO was added andincubated at 37ºC with 5% CO2 for 30 min and washedwith 2 mL of warm PBS. Finally the cells were observedunder fluorescence microscope (510-590 nm) (Coligan,et al., 1995).

Total No. of red cells% apoptotic cells= ------------------------------------------ X 100

Total No. of red and green cells

Statistical analysisThe data were subjected to one-way analysis of variance(ANOVA) to evaluate the significant of difference ofmeans of various treatment groups using SPSSstatistical software package (Version: 10). The valuesare presented as mean ± S.D and P


4/5



Different solvents (hexane, acetone, IPA) dried fruitextracts of H. isora were used for DPPH radicalscavenging activity. Compared to hexane and IPA, It wasfound that acetone extract (60 μg/mL) ofH. isora recorded strong radical scavenging activity of96.44±0.57% whereas, hexane and IPA showed lowantioxidant activity (Table 2). According to Suthar et al.(2009) the antioxidant activities measured in hot waterextract of H. isora showed IC50 value of 25.12±0.18

µg/mL, which was comparable to that of ascorbic acid(IC50 2.75±0.29 µg/mL).

Hexane, acetone, IPA and crude protein fruit extracts ofH. isora were used for cytotoxicity/cell viability againsthuman lung cancer cells. Acetone recorded 93.7% (10µg/mL) whereas IPA recorded 96.2% (40 µg/mL). Crudeprotein recorded 90.3% (40 µg/mL) and hexane extractrecorded 62.5% (40 µg/mL) respectively. All the extracts

showed good toxicity to the cancer cells. Among theextracts tested, acetone fruit extract showed excellentcytotoxicity against the cancer cells (Table 3; Fig. 3). Itwas evident from MTT assay that acetone extract hadthe best anticancer property as compared to the otherextracts supported by Mosmann (1983) in his study.Intracellular ROS was measured using a non-fluorescentprobe, 2,7,-diacetyl dicholorofluorescein diacetate(DCFH-DA) that can penetrate into the intracellularmatrix of cells where it is oxidized by ROS to fluorescentdichlorofluorescein (DCF). The non-fluorescent DCFH-DA is oxidized by intracellular ROS and forms the highlyfluorescent DCF (Philip Jesudason et al., 2008) which is

measured spectrofluorimetrically (Fig. 5, 6).

Measurement of Mitochondrial Membrane potential Alteration in mitochondrial membrane potential(depolarization) is an indication of early stages ofapoptosis. Rhodamine 123 (Rh 123) is lipophilic cationicdye, highly specific for mitochondria. Polarizedmitochondria are marked by orange red fluorescenceand depolarized mitochondria are marked by greenfluorescence (Fig. 7, 8). Apoptotic nuclei exhibitingtypical changes such as nuclear condensation andsegmentation were stained by AO/EB. H. isora extracts,both treated and untreated cells (2 x 10

4 /well) were

seeded into 6-well plate and incubated in CO2 incubator

for 24 h and then the apoptotic morphological changeswere observed using a fluorescence microscope underblue filter (Fig. 9).


5/5



The typical morphological features of apoptosis,including increased cell membrane permeability,cellular shrinkage and granulation in the nucleus, aremajor consequences of the apoptotic trigger. NCI-H460cells treated with selected best extracts exhibitedapoptotic morphological changes, as detected with

AO/EB double staining. The numbers of apoptotic cells,including early-apoptotic (AO+, EB–) and late-apoptoticcells (AO+, EB+) were increased in a dose-dependentmanner. After treatment with acridones, an increase incell membrane permeability was observed, as evidencedby the red fluorescence of EB in the nucleus. At lowconcentrations, a majority of AO+, EB– cells weredetected, indicating apoptotic granulation and an intactcell membrane. As the concentration was increased,late-apoptotic (AO+, EB+) and necrotic (AO–, EB+) cellpopulations appeared (Réthy et al., 2007). This assayhelped us to visualize the different stages of apoptosis.Both acetone extract and the crude protein exhibited

good anticancer activity.

Conclusion Antioxidant and anticancer activity was evaluated usingvarious solvent extracts (hexane, IPA and acetone) andcrude protein. Dot plot assay conformed presence ofantioxidant activity, acetone fruit extract of H. isora showed 96.44% strong antioxidant activity compared tohexane, and IPA. Acetone extract exhibited bettercytotoxicity against human lung cancer cells (NCI-H460)whereas; acetone and crude protein extracts showedactivity against ROS. The investigation revealed theantioxidant and anticancer activity of H. isora dried fruit

extracts. To conclude, further investigations arenecessary to find out the active bio active moleculesresponsible for inhibitory mechanisms for its anticancerand antioxidant activity.

AcknowledgementsThe authors are thankful to Dr. K.V. Pugalendhi, Headand Dr. N. Rajendra Pasad, Asst. Prof., Dept. ofBiochemistry and Biotechnology, Annamalai University, Annamalai Nagar, Chithambaram for providingnecessary laboratory facilities.

References1. Anonymous. 1997. The wealth of India, New Delhi:CSIR, 5: 27-29.

2. Arjun, P., Saranya Sivan, P.S., Mohana Priya, S.,Krishnamoorthy, M. and Balasubramanian, K. 2012.Phytochemical analysis and anticancer activity ofNelumbo nucifera extracts. J. Acad. Indus. Res. 1(2): 81-85.

3. Bapalal, V. 1982. Some Controversial drugs in Indianmedicine. Varanasi:Chaukhambha, pp8-17.

4. Bhosle, S.M., Huilgol, N.G. and Mishra, K.P. 2005.Enhancement of radiation-induced oxidative stress andcytototoxicity in tumor cells by ellagic acid. Clin. Chim. Acta. 359: 89-100.

5. Bradford, M.M. 1976. A dye binding assay for protein. Anal. Biochem. 72: 248-254.

6. Chang, W.C., Kim, S.C., Hwang, S.S., Choi, B.K., Ahn,H.J., Lee, M.Y., Park, S.H. and Kim, S.K. 2002. Antioxidant activity and free radical scavenging capacitybetween Korean medicinal plants and flavonoids byassay-guided comparison. Plant Sci. 163: 1161-1168.

7. Chopra, RN., Chopra, IC., Handa, KL. and Kapoor, LD.

1956. Glossary of medicinal Plants, New Delhi: CSIR,131.8. Coligan, J.E., Kruisbeek, A.M., Margulies, D.H.,

Shevach, E.M. and Strober, W. 1995. Currentprotocols in Immunology. In: Related isolationprocedures and functional assays. 1: p17.

9. Kirtikar, K.R. and Basu, B.D. 1993. Indian MedicinalPlants, 2

nd ed. Allahabad: Lalit Mohan Basu: 1: 371-372.

10. Mathew, P.J. and Unnithan, M.C. 1992. Search for planthaving anticancer properties used by the tribal ofWynadu, Malappuram and Palghat districts of Kerala,India. Aryavaidyan, 6(1): 54-60.

11. Mosmann, R. 1983. Rapid colorimetric assay for cellulargrowth and survival: application to proliferation and

cytotoxicity assay. J. Immunol. Method. 65: 55-63.12. Muthu kumar, T., Mary Violet Christy, A., Anusha

Mangadu, Malaisamy, M., Sivaraj, C., Arjun, P., RaamanN. and Balasubramanian, K. 2012. Anticancer andantioxidant activity of Curcuma zedoaria and Curcumaamada rhizome extracts. J. Acad. Indus. Res. 1(2): 91-96.

13. Nagarajan, S., Arjun, P., Raaman, N., Shah, S.,Elizabeth Sobhia, M. and Mohan Das, T.2012. Selectivesynthesis of sugar-based β-lactum derivatives: Dockingstudies and its biological evaluation. Tetrahydron. 68:3037-3045.

14. Philip, J., Gunasingh, M., Charles, E.J., Solomon, F.D.

and Paul, R. 2008. Efficacy of DL-a lipoic acid againstsystemic inflammation induced mice: Antioxidantdefense system. Mol.Cell Biochem. 313: 113-123.

15. Quinchun, Z., Chengbin Cui, Bing Cai, Feng Qui, TaoGuo and Yao Xinsheng. 2005. Cell cycle inhibitoryactivity of 137 Traditional Chinese herbal medicinesagainst FT210 cell line. Pharmaceu. Biol. 43(2):135-139.

16. Réthy, B., Zupkó, I., Minorics, R., Hohmann, J.,Ocsovszki, I. and Falkay, G. 2007. Investigation ofcytotoxic activity on human cancer cell lines of arborinineand furanoacridones isolated from Ruta graveolens.Planta Med. 73: 41-48.

17. Singh, S.B., Singh, A.K. and Thakur, R.S. 1984.Chemical constituents of the leaves of Helicteres isora.

Ind. J. Pharmaceu. Sci. 46(4): 148-149.18. Soler-Rivas, C., Espín, J.C. and Wichers, H.J. 2000. An

easy and fast test to compare total free radicalscavenger capacity of foodstuffs. Phytochem. Anal. 11:330-338.

19. Suthar, M., Rathore, GS. and Pareek, A. 2009. Antioxidant and antidiabetic activity of Helicteres isora (L.) fruits. Ind. J. Pharm Sci., 71(6): 695-699.

20. Yoganarasimhan, S.N. 1996. Medicinal Plants of India,Karnataka: Interline Publishing Private Limited, 1: 237.

10 muthukumar aug manuscript

Documents